Ulla Rüetschi

Enrichment of glycopeptides for glycan structure and attachment site identification

We present a method to enrich for glycoproteins from proteomic samples. Sialylated glycoproteins were selectively periodate-oxidized, captured on hydrazide beads, trypsinized and released by acid hydrolysis of sialic acid glycosidic bonds. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.

Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, is considered a critical step in the pathogenesis of Alzheimer’s disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ peptides and glycopeptides from CSF samples and then liquid chromatography—tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(−39, −21, −20, and −13), in a series of APP/AβX-15 glycopeptides, where X was −63, −57, −52, and −45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)1-2Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD.

Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF

This investigation describes the discovery of novel possible cerebrospinal fluid (CSF) biomarkers for frontotemporal dementia (FTD) using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Sixteen clinically diagnosed FTD patients and 12 non-demented controls were included in the study. CSF was collected and analyzed for protein expression by SELDI-TOF MS. The samples were analyzed on four different array surfaces using two different energy-absorbing molecules as matrices. In total each sample was subjected to eight different surface/matrix conditions. About 2000 protein peaks (mass/charge ratios) were detected. Forty-two peaks were differentially expressed in FTD (P < 0.01). After exclusion of peaks with low signal-to-noise ratio and/or poor resolution and peaks representing differentially charged proteins, 10 peaks remained, five of which were increased and five decreased in FTD cases compared to controls. Using partial least square discriminant analysis (PLS-DA), the combination of these biomarkers discriminated FTD from non-demented controls with a sensitivity of 94%, a specificity of 83% and an accuracy of 89%. Five of the peaks were purified further and identified by tandem MS as a fragment of neurosecretory protein VGF, transthyretin, S-cysteinylated transthyretin, truncated cystatin C and a fragment of chromogranin B. With use of these potential biomarkers, FTD can be distinguished from control subjects with high accuracy in this pilot study.

Clinical proteomics in neurodegenerative disorders.

Neurodegenerative disorders are characterized by neuronal impairment that eventually leads to neuronal death. In spite of the brain’s known capacity for regeneration, lost neurons are difficult to replace. Therefore, drugs aimed at inhibiting neurodegenerative processes are likely to be most effective if the treatment is initiated as early as possible. However, clinical manifestations in early disease stages are often numerous, subtle and difficult to diagnose. This is where biomarkers that specifically reflect onset of pathology, directly or indirectly, may have a profound impact on diagnosis making in the future. A triplet of biomarkers for Alzheimer’s disease (AD), total and hyperphosphorylated tau and the 42 amino acid isoform of β‐amyloid, has already been established for early detection of AD before the onset of dementia. However, more biomarkers are needed both for AD and for other neurodegenerative disorders, such as Parkinson’s disease, frontotemporal dementia and amyotrophic lateral sclerosis. This review provides an update on recent advances in clinical neuroproteomics, a biomarker discovery field that has expanded immensely during the last decade, and gives an overview of the most commonly used techniques and the major clinically relevant findings these techniques have lead to.

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS2, Hex5HexNAc4-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS3, of selected fragment-ions from the CID-MS2 analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where “intact” glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.

LC–MS/MS Characterization of O-Glycosylation Sites and Glycan Structures of Human Cerebrospinal Fluid Glycoproteins

The GalNAc O-glycosylation on Ser/Thr residues of extracellular proteins has not been well characterized from a proteomics perspective. We previously reported a sialic acid capture-and-release protocol to enrich tryptic N- and O-glycopeptides from human cerebrospinal fluid glycoproteins using nano-LC-ESI-MS/MS with collision-induced dissociation (CID) for glycopeptide characterization. Here, we have introduced peptide N-glycosidase F (PNGase F) pretreatment of CSF samples to remove the N-glycans facilitating the selective characterization of O-glycopeptides and enabling the use of an automated CID-MS(2)/MS(3) search protocol for glycopeptide identification. We used electron-capture and -transfer dissociation (ECD/ETD) to pinpoint the glycosylation site(s) of the glycopeptides, identified as predominantly core-1-like HexHexNAc-O- structure attached to one to four Ser/Thr residues. We characterized 106 O-glycosylations and found Pro residues preferentially in the n - 1, n + 1, and/or n + 3 positions in relation to the Ser/Thr attachment site (n). The characterization of glycans and glycosylation sites in glycoproteins from human clinical samples provides a basis for future studies addressing the biological and diagnostic importance of specific protein glycosylations in relation to human disease.

Novel markers of osteogenic and adipogenic differentiation of human bone marrow stromal cells identified using a quantitative proteomics approach

Today, the tool that is most commonly used to evaluate the osteogenic differentiation of bone marrow stromal cells (BMSCs) in vitro is the demonstration of the expression of multiple relevant markers, such as ALP, RUNX2 and OCN. However, as yet, there is no single surface marker or panel of markers which clearly defines human BMSCs (hBMSCs) differentiating towards the osteogenic lineage. The aim of this study was therefore to examine this issue. Stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was utilized to investigate differently expressed surface markers in osteogenically differentiated and undifferentiated hBMSCs. Labeled membrane proteins were analyzed by mass spectrometry (MS) and 52 proteins with an expression ratio above 2, between osteogenically differentiated and undifferentiated cells, were identified. Subsequent validation, by flow cytometry and ELISA, of the SILAC expression ratios for a number of these proteins and investigations of the lineage specificity of three candidate markers were performed. The surface markers, CD10 and CD92, demonstrated significantly increased expression in hBMSCs differentiated towards the osteogenic and adipogenic lineages. In addition, there was a slight increase in CD10 expression during chondrogenic differentiation. Furthermore, the expression of the intracellular protein, crystalline-αB (CRYaB), was only significantly increased in osteogenically differentiated hBMSCs and not affected during differentiation towards the chondrogenic or adipogenic lineages. It has been concluded from the present results that CD10 and CD92 are potential markers of osteogenic and adipogenic differentiation and that CRYaB is a potential novel osteogenic marker specifically expressed during the osteogenic differentiation of hBMSCs in vitro.

Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC–MS/MS of Glycopeptides

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.

An online nano‐LC‐ESI‐FTICR‐MS method for comprehensive characterization of endogenous fragments from amyloid β and amyloid precursor protein in human and cat cerebrospinal fluid

Amyloid precursor protein (APP) is the precursor protein to amyloid β (Aβ), the main constituent of senile plaques in Alzheimers disease (AD). Endogenous Aβ peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aβ peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aβ peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aβ processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aβ was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aβ (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aβ peptide species in CSF by using an online top-down MS-based method.

Proteomic profiling of cerebrospinal fluid in parkinsonian disorders

Parkinsons disease (PD) and atypical parkinsonian disorders (APD), including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), are a group of neurodegenerative diseases sharing many similar signs and symptoms but distinguished by their particular clinical features, treatment response, prognosis and mortality. The differential diagnosis may be challenging, especially in early disease stages. Considering the importance of an accurate diagnosis both for clinical management and for research, new diagnostic tools are needed. In this study, we investigated 56 PD, 42 MSA, 39 PSP, 9 CBD patients, and 24 healthy controls. After screening the cerebrospinal fluid (CSF) proteome using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), we identified 4 proteins (ubiquitin [mass-to-charge ratio (m/z) 8590], beta2-microglobulin [m/z 11730], and 2 secretogranin 1 [chromogranin B] fragments [m/z 7260 and m/z 6250]) that differentiated healthy controls and PD patients from patients with APD. However, they could not differentiate PD patients from controls. As none of these changes were APD subgroup-specific, they most likely reflect the intensity and/or extent of the neurodegenerative process in general.